US10457747B2 - Method for obtaining high-yield, stable expression cell clones and antibody molecules obtained thereby - Google Patents

Method for obtaining high-yield, stable expression cell clones and antibody molecules obtained thereby Download PDF

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US10457747B2
US10457747B2 US15/549,087 US201615549087A US10457747B2 US 10457747 B2 US10457747 B2 US 10457747B2 US 201615549087 A US201615549087 A US 201615549087A US 10457747 B2 US10457747 B2 US 10457747B2
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protein
cell lines
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clones
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MeyLen CHEA
Julio PALACIOS
Miguel Arias
Loany CALVO
Tamara GONZÁLEZ
Rolando PÉREZ
Zhi BAI
Yuemao LIU
Kaiheng XIAO
Xiao Chen
Zhenhua HE
Yangliu CAI
Zhenhua Yang
Xianhong Bai
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Biotech Pharmaceuticals Co Ltd
Centro de Immunologia Molecular
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/95—Protein-free medium and culture conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to biotechnology, specifically to an efficient method to select stable high producer cell clones to be used in perfusion fermentation processes in order to produce therapeutic antibodies.
  • Therapeutic antibodies constitute the major category of biopharmaceuticals into the marketplace (Walsh G, 2014, Nature Biotechnology 2014, 32: 992-1000).
  • Several therapeutic antibodies have obtained registration approval for the treatment of cancer, autoimmune diseases and other chronic diseases and dozens of recombinant antibodies are in different phases of clinical development (Biologic Medicines in Development, Phrma Report 2013, www.phrma.org).
  • Usually patients receive hundreds milligrams of therapeutic antibodies in each dose, therefore there is currently a huge demand of production capacity worldwide.
  • Perfusion fermentation-based production processes allow high density cell cultures and potentially high antibody concentration in fermentation harvest.
  • long-term high density cell culture requires stable high producer clones to really optimize antibody production.
  • Protein-free media have been developed for biopharmaceutical production, for example PFHMII cell culture medium (reference from Hyclone).
  • Recombinant antibody-producing NS0 cell clones have been successfully adapted to grow in protein-free media (WO 2004/038010 A1). However, adaptation to serum free-media and further long-term fermentation process in serum-free media are most usually accompanied by a loss of cell line productivity (Barnes et al., 2003, Biotechnol Bioeng 81: 631-639; Barnes et al., 2004, Biotechnol Bioeng 85: 115-121). Stable high producer cell clones adapted to grow in protein-free medium can be recovered from unstable recombinant myeloma cell lines for industrial use (CN104152415A).
  • Biopharmaceuticals in particular therapeutic antibodies, are complex glycoprotein molecules. Any change in the production process might introduce variations in product attributes. The concept of comparability has emerged to assess such variations in product attributes (Demonstration of comparability of human biological products, including therapeutic biotechnology-derived products, Center for Biologics Evaluation and Research (CBER), Center for Drug Evaluation and Research (CDER) April 1996. www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm122879.htm; EU Guideline on Comparability of Medicinal Products containing Biotechnology-derived Proteins as Active Substances: Quality issues (CPMP December 2003).
  • 14F7 is a monoclonal antibody (mAb) specific for the tumor associated antigen N-glycolyl-GM3 ganglioside [GM3(Neu5Gc)] (Patent number ZL 99800261.5; Carr et al., 2000, Hybridoma 19, 241-247).
  • mAb monoclonal antibody
  • 14F7 mAb is able to kill tumor cells expressing the ganglioside in a complement-independent manner (Carr et al., 2002, Hybrid Hybridomics 21, 463-468; Roque-Navarro et al., 2008, Mol Cancer Ther 7, 2033-2041).
  • a humanized version of this antibody obtained by the modification of potential human T cell epitopes (Patent application No. 200480017457.8; Mateo et al., 2000, Hybridoma 19, 463-471) and named 14F7h, retained the properties of the mouse and chimeric antibodies (Fernandez-Marrero et al., 2011, Immunobiology 216, 1239-1247).
  • Humanized 14F7hmAb is of potential value for the therapy of GM3(NeuGc)-expressing tumors.
  • GM3(NeuGc)-expressing tumors [0010]
  • NS0 myeloma cell lines expressing antibody 14F7h lost viability in high density cell cultures because these cells expressed the antigen GM3(Neu5Gc) and were killed by the secreted cytotoxic antibody.
  • the recombinant humanized antibody 14F7h was expressed in murine NS0 myeloma cell line defective in N-glycolylated-glycoconjugates, due to the knock down of the CMP-N-acetylneuraminic acid hydroxylase enzyme (Fernandez-Marrero et al., 2011, Immunobiology 216, 1239-1247).
  • Such cell line showed to be very difficult to adapt to grow in serum-free medium.
  • This procedure comprises recombinant myeloma cell lines for which the process of adaptation from serum-free medium to protein-free medium is not possible or takes long-time and is accompanied by the loss of antibody production due to the emergence of non-producer cell population.
  • Key innovative step in the present invention is the adaptation process to grow in protein-free medium in high cell density (8-10 ⁇ 10 6 cells/ml) which increases the frequency of stable high producer cell clones.
  • the method of the present invention consists of three stages process:
  • the first stage was performed by defrosting the cell line in protein-free cell culture medium (PFHMII) supplemented with 3-4 g/L of Cell Boost (enriched in cholesterol, lipids and other nutrients). Maximal cell concentration (Xv) was ranging from 0.9-1.2 ⁇ 10 6 cell/ml. Serial passage was performed until Xv reached a constant value. Initial cellular concentration after each passage was adjusted to 0.4-0.5 ⁇ 10 6 cell/ml. After seven passages Cell Boost supplement was reduced to 1-2 g/L, and after four additional passages Cell Boost supplement was completely removed. Then cells were allowed to grow in protein-free medium without any supplement for more than 60 days. Xv reached values between 1.5-1.8 ⁇ 10 6 cell/ml with around 95% cell viability.
  • stable high producer cell clones from cells at the end of fermentation were selected.
  • Defrosted cells from EPCB were subjected to cell cloning by the limiting dilution method (Freshney R., 2010, Culture of animal cells: a manual of basic technique and specialized applications (6th ed.). Hoboken, N.J.: Wiley-Blackwell. pp. 208-211).
  • Secreted antibody in cell culture supernatant was measured by an ELISA method using the anti-14F7h MAb anti-idiotypic antibody 4G9 as capture antigen. Cell culture supernatants were diluted 1/500.
  • Clones yielding absorbance values above the median one were selected for further characterization by Flow Cytometry (FACS), Intracellular immunoglobulin staining was performed using an anti-human IgG antibody conjugated with FITC and measured by FACS (Pluschke et al., 2011, BMC Proceedings 5, Suppl 8:P97). Mean Fluorescence Intensity (MFI) and percentage of positive cells were determined for each isolated clone. Surprisingly more than 80% of evaluated clones showed a predominant antibody-producer subpopulation.
  • FACS Flow Cytometry
  • Clones having more than 95% antibody-producer subpopulation and higher MFI were selected for kinetic studies in spinner flasks and 5 L bioreactor to evaluate cell concentration (Xv), cell viability (%), specific growth rate (p) and IgG concentration in cell culture supernatant.
  • Cell concentration was found between 4-5 ⁇ 10 6 cells/ml.
  • Cell viability during the exponential growth phase was about 85%.
  • Specific growth rate varied between 0.015-0.025 h ⁇ 1 .
  • IgG concentration was found between 70-120 mg/L.
  • long-term stability of stable high producer cell clones was evaluated by performing kinetic studies after 30, 60 and 90 days in continuous cell culture. Specific growth rate (p) and specific production rate (qp) were determined.
  • identity attributes of the secreted immunoglobulin by selected stable high producer cell clones were determined. Disclosure of such identity attributes operationally defines the molecular phenotype of this therapeutic antibody 14F7h:
  • FIG. 1 Cell adaptation to PFHM-II medium at different concentrations of Cell Boost 5.
  • FIG. 2 Fermentation run in 5 L bioreactor using the cells adapted to PFHM-II medium without cell boost 5.
  • FIG. 3 Absorbance values from the recognition ELISA. Black line is the median of the results. Purified 1 ⁇ g/mL of T1h and hR3 were used as negative controls. Selected clones 35D8, 35D6, 31E7, 31C7, 23E9, 23C2, 73F4, 73E5, 72G5, 72F6, 72D3 (gray color), discarded clones 35F7, 33E8, 23E4, 22D8, 21E6, 2105, 73C4, 72D9, 71F2, 62E7, 61E3 (black color).
  • FIG. 4 Selected cells were analyzed by flow cytometry and the percentage of high producer cell sub-population was determined. NS0 myeloma cell line was used as negative control and recombinant NS0 myeloma cell line expressing hR3 antibody was used as positive control. Selected clones 31E7, 23E9, 33E8, 35D6, 23C2, 72F6, 73F4, 72G5 (gray color), discarded clones 72D3, 73E5 (black color).
  • FIG. 5 MFI value of high producer cell sub-population was determined.
  • the myeloma cell line expressing hR3 and T1h antibodies were used as positive controls (light gray color).
  • Selected clones 31E7, 35D6, 72G5 (gray color); discarded clones 23E9, 33E8, 72D3, 73E5, 23C2, 72F6, 73F4 (black color).
  • FIG. 6 Growth curves of the evaluated clones, cell viability and integral of viable cells from kinetic studies in roller bottles and 5 L bioreactor are plotted (y axis) versus time (x axis).
  • FIG. 7 Comparative analysis of maximum specific growth rate and maximum IgG concentrations between selected clones growing in roller bottles and 5 L fermenter.
  • FIG. 8 Recognition of NeuGcGM3 ganglioside by 14F7h antibody produced by different selected clones. Supernatants were taken from 5 L bioreactor. The anti-EGFR antibody was used as negative control, 14F7h antibody produced by parental cell was used as positive control.
  • FIG. 9 IgG intracellular concentration measured by flow cytometry at different steps of the selection process.
  • FIG. 10 Comparative kinetic studies between parental cells and clone 31E7 performed in 5 L bioreactor.
  • Xv viable cell concentration
  • SXv integral of viable cells
  • IgG maximum antibody concentration.
  • FIG. 11 Kinetic studies to evaluate the stability of clone 31E7 were performed at 30, 60 and 90 days after cell thawing.
  • FIG. 12 IgG intracellular content measured by flow cytometry in samples from the stability study of clone 31E7.
  • FIG. 13 Deconvoluted mass spectrum of 14F7h Light Chain in its native (upper panel) and deglycosylated (lower panel; using PNGase F) states for the samples being reduced/alkylated.
  • Conventional LC-MS conditions were used with a C8 column for sample separation/desalting and Acetonitrile/Formic acid buffer system for running. Inserted figure corresponds to magnification of main peak region.
  • FIG. 14 Deconvoluted mass spectrum of 14F7h Heavy Chain in its native (upper panel) and deglycosylated (lower panel; using PNGase F) states for the samples being reduced/alkylated.
  • Conventional LC-MS conditions were used with a C8 column for sample separation/desalting and Acetonitrile/Formic acid buffer system for running. Inserted figure corresponds to magnification of main peak region.
  • FIG. 15 Deconvoluted mass spectrum of 14F7h Whole Molecule in its native (upper panel) and deglycosylated (lower panel; using PNGase F) states. Conventional LC-MS conditions were used with a C8 column for sample separation/desalting and Acetonitrile/Formic acid buffer system for running. Inserted figure corresponds to magnification of main peak region.
  • FIG. 16 Peptide mapping profile of 14F7h obtained after trypsin digestion and reverse phase HPLC separation using a C4 column and a conventional Acetonitrile/TFA buffer system.
  • FIG. 17 Circular Dichroism spectral analysis of 14F7 in the Far UV (205-260 nm) region obtained at 25° C. using a 2 mm path length cuvette. The spectra was obtained with a sample concentration of 0.6 mg/mL.
  • FIG. 18 Fluorescence emission of Tryptophan, using a Varioskan Flash equipment, read between 310 and 390 nm and obtained after exciting 14F7h molecule at 280 nm. A 96 well plate format was used, with 200 uL per well and a sample concentration of 0.2 mg/mL.
  • FIG. 19 Glycosylation profiling of 2-AB labeled glycans isolated from 14F7h and separated using normal phase HPLC with fluorescent detection (Ex: 330 nm/Em: 420 nm). Squared broken lines show a magnification of the minor peaks.
  • FIG. 21 Weak cation exchange profile of 14F7h using a Propac-WCX10 column, obtained at 280 nm. 30 ug of sample were injected.
  • FIG. 22 SEC-HPLC profile of 14F7 using a TSK-G3000sxl column, obtained at 280 nm.
  • FIG. 23 Flow cytometer dose-response curve (% positive vs log of 14F7h concentration (ug/mL)) using L1210 target cells for different samples of antibody tested on different days.
  • FIG. 24 Cytotoxic effect induced by 14F7h mAb in X63 mouse myeloma cells.
  • A Binding properties of 14F7h mAb. X63 cells were stained with 10 ⁇ g/mL of the antibody followed by a FITC-conjugated rabbit anti-human IgG antibody. Herceptin mAb (anti-human Her-2) was used as negative control.
  • B X63 cells were treated with 100 ⁇ g/mL of 14F7h mAb. Cell viability after 6 h incubation at 37° C. was evaluated by propidium iodide (PI) uptake and flow cytometry analysis. Cytotoxicity is expressed as percentage of PI-stained cells. Herceptin mAb was used as negative control.
  • PI propidium iodide
  • FIG. 25 In vivo anti-tumor effect of 14F7h mAb on a mouse myeloma model.
  • A Schedule of mAb administration. X63 mouse myeloma cells (0.2 ⁇ 10 6 ) were inoculated subcutaneously at day 0 to BALB/c mice and the antibodies were administered intravenously (300 ⁇ g) at days 2 to 5.
  • B Kaplan-Meier curves of tumor-free survival up to day 106. Humanized T1h mAb (anti-human CD6) was used as negative control. Statistical analysis was performed with the log-rank test.
  • Example 1 Adaptation to Protein-Free Medium by a Stepwise Reduction of a Lipid-Enriched Supplement to Chemically Defined Medium in Low Density Stationary Cell Culture
  • the cell line 14F7htb58 was adapted to growth in PFHMII cell culture medium without supplement cell boost 5.
  • the process of reduction of supplement cell boost 5 was performed in 75 cm 2 T flasks, in stirred shaker (80 rpm) at 36.5° C. and 5% CO 2 . Every 48-72 hours the concentration of viable cells and the percentage of cell viability were determined. Cell concentration was adjusted to 0.4-0.5 ⁇ 10 6 cell/ml in every culture passage.
  • Frozen 14F7htb58 cells were thawed in PFHMII supplemented with 3.5 g/L cell boost 5. After thawing cell viability was 75%, and then it increased up to over 90% ( FIG. 1 ).
  • serial culture passages were performed to maintain a maximal concentration of viable cells in the range of 0.9-1.8 ⁇ 10 6 cell/ml, with percentages of cell viability higher than 90%.
  • concentration of cell boost 5 was reduced to zero, with 95% of cell viability and 1.2-1.8 ⁇ 10 6 cell/ml.
  • the adaptation process to PFHMII cell culture medium without lipids and cholesterol took 60 days approximately.
  • 14F7htb58 cells adapted to growth in protein-free PFHMII cell culture medium were inoculated in a 5 L fermenter at a concentration of 0.4-0.5 ⁇ 10 6 cell/ml. Fermenter operation parameters were: pH 6.9-7.0; 105 RPM; 40% dissolved oxygen; 36.5-37.0° C.; work volume 3.5 L. Cell viability and cell concentration were monitored daily by Trypan Blue exclusion method (Sigma) using a Neubauer chamber. Fermenter operation in perfusion mode was performed after cell concentration reached the value of 2.5 ⁇ 10 6 cell/ml. A hollow fiber cartridge was employed for the perfusion mode and a dilution rate of 0.3-0.7 VVD was used.
  • Thawed cells from EPC were cloned to a single cell per well in 96 well plates using the limiting dilution method and DMEM-F12 1:1 cell culture medium supplemented with 5-10% of fetal bovine serum. Culture plates were incubated at 36.5° C. in 5% CO 2 atmosphere. Cloning efficiency was below 2%. Twenty days after cell cloning culture supernatants were taken to assess IgG concentration by sandwich-type ELISA. The anti-idiotypic antibody 4G9 (3 ug/ml) was used as capture antigen and an anti-human heavy chain goat antibody coupled to alkaline phosphatase as a probe. All samples were diluted 1/500, and the median of supernatant absorbance was determined.
  • Clones with absorbance values higher than the median value were selected (23C2, 23E9, 31C7, 31E7, 35D6, 35D8, 72D3, 72F6, 72G5, 73E5, 73F4) ( FIG. 3 ). Selected clones were expanded to 24 well plates in DMEM-F12 1:1 cell culture medium supplemented with 5-10% of fetal bovine serum. Further cell expansion in T culture flasks were performed using PFHMII cell culture medium, in stirred shaker (80 rpm) at 36.5° C. and 5% CO 2 .
  • Intracellular IgG content was determined by flow cytometry (FACS) in selected clones.
  • FITC fluorescein iso-thiocyanate
  • To determine labeling percentage 4 ⁇ 10 5 cell/sample were analyzed. Clones having more than 95% of high producer cell sub-population were selected ( FIG. 4 ).
  • Another selection criterion was the normalized Fluorescence Median Intensity (FMI) and clones 35D6, 72G5 and 31E7 were selected ( FIG. 5 ).
  • FMI Fluorescence Median Intensity
  • Clone 31E7 showed the highest growth specific rate (>0.025 h ⁇ 1 ) both in roller bottles and 5 L bioreactor. Antibody concentration between 50-70 mg/L was found for all clones in 5 L bioreactor however clone 31E7 showed the highest antibody concentration in roller bottles ( FIGS. 7A and 7B ).
  • Samples were taken from the kinetic studies performed in 5 L bioreactor to evaluate the biological activity of the secreted antibody.
  • a sandwich-type ELISA was performed. Polysorp plates were coated with NeuGcGM3 ganglioside solution in methanol (10 ⁇ g/ml). As secondary antibody an anti-human heavy chain goat antibody coupled to alkaline phosphatase was used. All samples were adjusted to 1 ⁇ g/ml antibody concentration and diluted 1/10 in the ELISA test. All tested samples showed recognition of the ganglioside antigen, while clones 31E7 and 35D6 showed values between 70-80% with respect to the positive control ( FIG. 8 ).
  • the intracellular IgG content evolved along the process of phenotypic adaptation and cloning.
  • Parental cells 14F7htB58 growing in PFHMII plus Cell Boost 5 showed a bimodal distribution because of the existence of a non-producer cell sub-population.
  • 14F7htB58 cells were enriched in the producer cell sub-population (unimodal distribution), however for clone 31E7 a narrower single peak was obtained suggesting a more homogeneous cell sub-population ( FIG. 9 ).
  • clone 31E7 has higher maximal cell concentration, integral of viable cells and antibody production rate than parental cells ( FIG. 10 )
  • the stability of antibody production by clone 31E7 was evaluated during 90 days in cell culture. Kinetic studies in roller bottles were performed at 30, 60 and 90 days after cell thawing. Samples were taken to carry out flow cytometry studies. No differences were found between cells having different times in cell culture. Maximal cell concentration varied from 3.5-4.5 ⁇ 10 6 cell/ml, cell viability was higher than 90% and the antibody concentration ranged from 50-80 mg/L ( FIG. 10 ). The growth specific rate ( ⁇ ) kept higher than 0.025 h ⁇ 1 while the production specific rate (QP) was higher than 0.18 pg/cell*h ( FIG. 11 ). After 90 days in cell culture clone 31E7 showed a narrow single peak at same FMI representative of a homogeneous high producer cell population ( FIG. 12 ).
  • identity attributes are defined to cover the basic molecular properties that later will allow to assess quality of the molecule and to monitor product consistency as well as cell line stability.
  • Primary structure is studied by determination of the mass of the whole molecule, and its individual chains, by LC-ESI-MS analysis of the native and disulfide bridges reduced/alkylated sample (glycosylated and deglycosylated). See Table 1 for a summary of the results and FIGS. 13, 14, 15 .
  • the peptide mapping of the molecule is used to monitor its first order structure and further exclude any possible post-translational modification truncation or sequence changes ( FIG. 16 ).
  • FIG. 18 shows the results obtained for this latter test. In this case, emission maximum was obtained at 333 nm and the value of Absorbance Ratio 330 nm/350 nm was 1.23.
  • FIG. 19 shows the results obtained for three different samples, and their glycosylation parameters are shown in Table 2 (Montesino et al, 2012, Biologicals 40:288-298). The behavior and dispersion of these glycosylation parameters are shown in FIG. 20 .
  • the heterogeneity of the molecule is defined by two orthogonal methods.
  • Weak Cation Exchange (WCX) is used to detect the different charged species, allowing detecting product truncations, deamidations, some glycosylation variants, etc.
  • results of this method mainly monitor C-terminal lysine truncation, a common modification found in hlgG1 molecules (Dionex Application Note 127, http://www.dionex-france.com/library/literature/application_notes_updates/AN127_LPN1047.pdf).
  • the profiles obtained for different samples are shown in FIG. 21 and Table 3 shows the result of their integration. Additionally, Size Exclusion Chromatography (SEC) is used to monitor the aggregation state of the molecule.
  • FIG. 22 shows the profiles obtained from different samples and Table 4 shows the result of their analyses.
  • FIG. 23 shows the dose-response curve results obtained using different samples, and Table 5 shows the EC 50 calculated from them.
  • Antigen expression by X63 mouse myeloma cells was measured by flow cytometry. 100% of X63 mouse myeloma cells were stained by 14F7h mAb ( FIG. 24A ). X63 cells were incubated with 10 ⁇ g/mL of the antibody followed by a FITC-conjugated rabbit anti-human IgG antibody. Herceptin mAb (anti-human Her-2) was used as negative control.

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